Homogeneous Aptasensor with Electrochemical and Electrochemiluminescence Dual Detection Channels Enabled by Nanochannel-Based Probe Enrichment and DNase I Cleavage for Tumor Biomarker Detection

Homogeneous aptasensors that eliminate the need for probe labeling or immobilization hold significant potential for the rapid detection of tumor biomarkers. Herein, a homogeneous aptasensor with electrochemical (EC) and electrochemiluminescence (ECL) dual detection channels was developed by integrating nanochannel-based probe enrichment and DNase I cleavage for selective detection of the tumor biomarker, carbohydrate antigen 125 (CA125). A two-dimensional (2D) composite probe was prepared by assembling the CA125-specific aptamer and the cationic probe tris(2,2'-bipyridyl)Ru(II) (Ru(bpy)₃²⁺), which exhibited both EC and ECL properties, onto graphene oxide (GO) nanosheets (Ru(bpy)₃²⁺/Apt@GO). A vertically ordered mesoporous silica film (VMSF) with ultrasmall, uniform, and vertically aligned nanochannel arrays was rapidly grown on the inexpensive and disposable indium tin oxide (ITO) electrode, forming the detection interface. Due to the size exclusion effect of the ultrasmall nanochannels in VMSF, the Ru(bpy)₃²⁺/Apt@GO probe was unable to penetrate the nanochannels, resulting in no detectable Ru(bpy)₃²⁺ signal on the electrode. Upon specific recognition of CA125 by the aptamer, an aptamer-CA125 complex was formed and subsequently detached from GO. DNase I then cleaved the aptamer-CA125 complex, releasing CA125 and allowing Ru(bpy)₃²⁺ to dissociate into the solution. This enzymatic cleavage enabled CA125 to re-enter the binding cycle, amplifying the release of Ru(bpy)₃²⁺ into the solution. The electrostatic adsorption of the cationic Ru(bpy)₃²⁺ by VMSF significantly enhanced both the EC and ECL signals. The constructed aptasensor exhibited a linear EC detection range for CA125 from 0.1 U/mL to 100 ng/mL, with a limit of detection (LOD) of 91 mU/mL. For ECL detection, CA125 was detected over a range from 0.001 to 100 U/mL, with a LOD as low as 0.4 mU/mL. The developed aptasensor demonstrated excellent selectivity and was successfully applied to the dual-mode EC/ECL detection of CA125 in fetal bovine serum samples.